P1347: H3K36 HISTONE METHYLTRANSFERASE NSD3 FUNCTIONS AS A CRUCIAL EPIGENETIC REGULATOR OF ERYTHROPOIESIS
Authors: Arunim Shah, Shobhita Katiyar, Khaliqur Rahman, Naresh Tripathi, Chandra Prakash Chaturvedi
Journal: HemaSphere (2023)

Topic: 23. Hematopoiesis, stem cells and microenvironment Background: Histone methyltransferase NSD3 methylates histone 3 at lysine 36, leading to active gene transcription. NSD3 is found to be deregulated in various cancers, including hematological malignancies. However, the role of NSD3 in regulating normal hematopoietic differentiation to myeloid and lymphoid lineages is not clearly understood. Aims: To elucidate the role of NSD3 in regulating erythropoiesis Methods: CD34+ Hematopoietic stem cells (HSCs) were positively isolated using MACS from the leukapheresis of a healthy donor. Cells were cultured in a serum-free medium containing BIT serum substitute in the presence of EPO (5IU/ml), IL-3 (10 ng/ml), and stem cell factor (SCF; 100 ng/ml) as described. Lentiviral mediated NSD3 knockdown (KD) was done on days 4, 8, and 12 of erythroid differentiation. The effect of NSD3 KD on erythroid differentiation was studied using the panel of cell surface markers by FACS after 72 hrs post KD. The gene expression of erythroid-specific transcription factors and heme biosynthetic pathway genes were observed by q-RT PCR. While cell proliferation, cell viability, and apoptosis were assessed by Click-IT Edu and Annexin/Propidium staining. Results: The knockdown of NSD3 leads to aberrant erythroid differentiation in HSCs. We observed that various progenitor stem cell populations were altered upon NSD3 knockdown on days 4, 8, and 12 of ex-vivo erythroid differentiation. The KD of NSD3 on day 4 led to a 60% decrease in the megakaryocytes erythroid progenitor population. Similarly, a significant reduction in BFU-e and CFU-e colonies in methocult assay was observed upon NSD3 KD. FACS analysis of BFU-e and CFU-e revealed an 80% reduction in the BFU-e population and a 20% reduction in the CFU-e population with significant CD36/CD71/GPA reduction erythroid differentiation markers. Furthermore, KD of NSD3 leads to a block in the terminal erythroid differentiation by aberrantly increasing the basophilic erythroblasts and decreasing ortho/polychromatic erythrocytes. At the molecular level, we found that the knockdown of NSD3 leads to the downregulation of the b-globin gene, which was accompanied by significant downregulation of EKLF, one of the crucial transcription factors of erythroid differentiation of erythroid-specific factor. We also observed that other erythroid-specific transcription factors, viz. GATA1, FOG1, and NF-E2 were also affected. Furthermore, we observed a significant downregulation of ALAS2, a key regulator of heme biosynthesis on NSD3 KD, while other heme biosynthetic genes (ALAS1, PBGD, FECH) were also affected, suggesting NSD3 may be a crucial epigenetic regulator that controls and regulates erythropoiesis. We further found KD of NSD3 induces apoptosis, where live cells decrease, and early apoptotic cells increase. We also found the population of proliferating CD36/CD71 significantly reduced upon NSD3 KD. Summary/Conclusion: The mechanistic basis of NSD3-mediated carcinogenesis is robustly studied. However, its role in regulating various cellular and biological processes, including normal hematopoiesis, remains unexplored. Here we have studied the role of NSD3 in the regulation of erythropoiesis. Depleting NSD3 leads to decreased proliferation and defects at the early multipotent progenitors, CFU-E/-BFU-E progenitors, and cells associated with the terminal differentiation stage of hematopoietic stem cells differentiation into erythroid cells. At a molecular level, NSD3 regulates the different stages of erythroid differentiation by controlling the transcription factors associated with the adult b-globin gene regulation and genes involved in heme biosynthesis. Overall, our study reveals a previously undescribed role of that NSD3 in regulating human erythropoiesis.Keywords: Erythropoieisis